The long-term objective of this proposal is to establish the structure- activity relationships of phospholipase A2 (PLA2) through the determination of the x-ray structure of its mutants and their complexes with the transition state analogue, diC8(2Ph)PE. This will be carried out in collaboration with Ming-Daw Tsai who has cloned and over expressed the enzyme and is studying the kinetics and solution structure by NMR and Michael H. Gelb who will be supplying the transition state analogue. Although, the x-ray structures of PLA2 from mammalian pancreas, snake and bee venom, synovial fluids and their complexes with the transition state analogues are known, there still are major gaps in the PLA2 structural field. For example, the absence of a systematic study of the effects of mutations on the catalytic site, lipid binding loci and interfacial recognition surface are lacking. Also there is no structural information on the effect of mutations on the binding of substrate analogues. This proposal seeks to bridge these gaps. It is planned to carry out the x- ray structures of the (i) mutants of the catalytic site (H48N, D99N, Y52F, F22X, F106X and Y69X), (ii) the mutants of the hydrophobic channel (L2X, F5X, I9X, L19X), (iii) complexes of the mutants with the transition state analogue, diC8(2Ph)PE, (iv) the mutants of the important disulfide bridges where the Cys/Cys pair is changed to Ala/Ala and (v) the mutants of the N-terminal Ala-1 for its effect on the stability of the catalytic pocket and its role in the interfacial recognition. To date the structures of the wild type (1.8 alpha), the single mutant K56M (1.8 alpha) and the double mutant Y52F/Y73F (1.93 alpha) have been solved in our laboratory. The K56M structure has indicated a possible head-group binding site for the phospholipids. The double mutant Y52F/Y73F has unequivocally established that the phenolic groups of the highly conserved Tyr residues (52 and 73) are not replaced by water molecules in the active site, and further demonstrated that the hydrogen bonding by the phenolic groups are not essential for supporting the catalytic pocket. This work is health related in that it will be useful in rational design of more potent inhibitors of PLA2 which have therapeutic applications.